All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral-based systems for gene transfer purposes have been described, such as retroviral systems, which are currently the most widely used viral vector systems for gene transfer. For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109. However, the recent description of retrovirus vector-associated leukemogenesis in two patients has underscored potential limitations of this vector system.
A number of adenovirus-based gene delivery systems have also been developed. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range both in vivo and in vitro. Adenovirus is easily produced at high titers and is stable so that it can be purified and stored. For descriptions of various adenovirus-based gene delivery systems, see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476. However adenovirus virus vectors, including the newer helper-dependant adenovirus vectors are associated with triggering host innate immunity that can be highly toxic.
Eukaryotic vectors based upon the nonpathogenic parvovirus, adeno-associated virus (“AAV”), have recently emerged as promising vehicles for efficient gene transfer (Muzyczka, N., “Use of adeno-associated virus as a general transduction vector for mammalian cells,” Curr Top Microbiol Immunol, Vol. 158, pp. 97-129 (1992)). AAV is a replication-defective DNA virus with a 4.7 kb genome with palindromic inverted terminal repeats (“ITR”). Coinfection with a helper virus, typically adenovirus or herpes simplex virus, is required for productive infection. In the absence of helper virus coinfection, AAV stably integrates via the ITRs into chromosomal DNA, or may persist in an episomal state. Wild type AAV is unique in the capacity for integration into a specific region of human DNA termed “AAVS1” on human chromosome 19.
AAV have been found in many animal species, including nonhuman primates, canines, fowl, and humans (Murphy, F. A. et al., “Classification and nomenclature of viruses: sixth report of the International Committee on Taxonomy of Viruses,” Arch. Virol., Vol. 1995, pp. 169-175 (1995)). There are more than 100 serotypes of AAV, including AAV type 1 (AAV-1), isolated from primates, AAV-2, AAV-3, and AAV-5, isolated from humans, and AAV-6, isolated from a human adenovirus preparation; other serotypes are being intensively evaluated for use in gene therapy. See, e.g. Gao, P. (2004) J Virol. 78(12):6381-6388. AAV-2 is the most characterized primate serotype, since its infectious clone was the first one made (Samulski, R. J. et al., “Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells,” Proc. Natl. Acad. Sci. USA, Vol. 79, pp. 2077-2081 (1982)). The full sequences for AAV-3A, AAV-3B, AAV-4, and AAV-6 recently were determined (Chiorini, J. A. et al., “Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles,” J. Virol., Vol. 71, pp. 6823-6833 (1997); Muramatsu, S. et al., “Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3,” Virology, Vol. 221, pp. 208-217 (1996); and Rutledge, E. A. et al., “Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2,” J. Virol., Vol. 72, pp. 309-319 (1998)). Generally, all primate AAV show more than 80% homology in nucleotide sequence. AAV vectors have been based primarily on serotype 2, a human-derived parvovirus (Parks, W. P. et al., “Seroepidemiological and ecological studies of the adenovirus-associated satellite viruses,” J. Virol., Vol. 2, pp. 716-722 (1970); and Samulski, R. J., “Adeno-associated virus: integration at a specific chromosomal locus,” Curr. Opin. Genet. Dev., Vol. 3, pp. 74-80 (1993)).
The early availability of an infectious clone of AAV-2 stimulated work on the development of replication-defective vectors. The AAV2 genome has two major open reading frames (“ORFs”); the left encodes functions necessary for AAV ori mediated replication and site specific integration (Rep), while the right encodes functions necessary for encapsidation (Cap). AAV vectors transduce many different types of cells. Multiple studies have amply demonstrated that rAAV vectors can transduce quiescent, nonproliferating targets. rAAV vectors do not encode any viral encoded genes, reducing their intrinsic immunogenicity. In addition, prolonged in vivo transgene expression following rAAV transduction has been documented in animal models. Finally, since its discovery in the mid-1960s, wild type AAV has yet to be definitively identified as a pathogen in either animals or humans.
The construction of recombinant adeno-associated virus (“rAAV”) vectors has been described. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Patent Publication Numbers WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Gao, G. (2002) Proc Natl Acad Sci USA 99:11854-11859; Hauck, B. (2003) Journal of Virology 77(4):2768-2774; and Gao, G. (2004) Journal of Virology 78(12):6381-6388.
Multiple schwannomas in peripheral distal and intracranial nerves are the hallmark of neurofibromatosis 1 and 2 (NF1 and NF2), and schwannomatosis, three types of nerve sheath tumors, classified as neurocutaneous syndromes, with incidences of about 1 in 3,200, 1 in 32,000 and 1 in 1,000,000, respectively (Antinheimo et al., 2000; Baser et al., 2006). Schwannomas are benign tumors composed of neoplastic dedifferentiated Schwann cells. Although typically nonmalignant and slow growing, these tumors can have devastating consequences for patients. They can cause extreme pain and compromise sensory/motor functions, including hearing and vision. Schwannomas in NF2 are frequently associated with neurological deficits, such as paresthesias, weakness, or hearing loss, and similar tumors in schwannomatosis often cause excruciating pain (Huang et al., 2004; Lu-Emerson and Plotkin, 2009). Some schwannomas become very large, causing compression of adjacent organs or structures, and can lead to paralysis or death due to progressive spinal cord or brainstem compression. Schwannomas may arise sporadically, without presenting any genetic features of NF1, NF2 and schwannomatosis. Most of vestibular schwannomas are sporadic schwannomas, so their incidence is very significant. Vestibular schwannomas usually occur as single tumors, not as multiple tumors throughout the body.
The underlying molecular abnormality in NF2 is a germline mutation of the NF2 gene. Somatic loss of the normal remaining NF2 allele in Schwann cells leads to deregulated growth of neoplastic Schwann cells with schwannoma formation (Rouleau et al., 1993). The timing of loss of the second wild-type allele may occur during development, as Schwann cells move out along axons and begin myelination, or in response to injury when Schwann cells dedifferentiate and commence proliferation (Jessen and Mirsky, 2005; McClatchey and Giovannini, 2005). In schwannomatosis some patients have a germline mutation in the SMARCB/IN11 gene with a second hit in Schwann cells leading to schwannoma formation (Hulsebos et al., 2007); in addition, the majority of these tumors harbor additional mutations in the NF2 gene (Jacoby et al., 1997; Hadfield et al., 2008). NF1, NF2 and schwannomatosis have three different genetic mutations, NF1, NF2 and INI, whereas no specific genetic mutation has been identified for sporadic schwannomas. The table below summarizes the features of these diseases that are all composed of Schwann cells and therefore amenable to various treatment methods described herein. In addition, their standard of care (as detailed below) is the same, and they all present the same need for new therapies.
USA Cell Genetic DiseasePrevalencecompositionMutationNeurofibromatosis100,000Schwann &NF1type 1fibroblastsSporadic 30,000SchwannUnknownSchwannomaNeurofibromatosis10,000SchwannMerlin (NF2)type 2Schwannomatosis6,000SchwannINI 1
The standard of care for patients with NF2 and schwannomatosis is surgical resection or radiosurgery of symptomatic tumors to reduce tumor size. Unlike in the case of sporadic schwannomas, in which typically only a single tumor is present and surgery is generally an efficacious treatment strategy as long as the lesion is accessible for resection (Lu-Emerson and Plotkin, 2009), in schwannomatosis and NF2, which present with multiple tumors, resection is confounded by both the inaccessibility of many tumors and by risk of nerve damage, including major motor dysfunction, significant sensory loss (including deafness in the case of NF2 vestibular schwannomas), and neuropathic pain. Thus, for most individuals there is substantial morbidity associated with schwannomas in both NF2 and schwannomatosis, as well as with the current therapies. This suffering and debility, in combination with the paucity of therapeutic options, makes the treatment of schwannomas a major unmet medical need.
Of the caspases family of genes, caspases-3 is the most common target for therapeutic modulation. Unlike caspases-3, caspases-1 has a strong pro-inflammatory component in addition to induction of apoptosis. Caspase-1 activates pro-IL-1b and pro-IL-18, which in turn trigger immune responses mediated by neutrophils and monocytes, or NK cells. In addition, caspases-1 has been associated with both innate and adaptive immunity and is activated by several chemotherapeutic drugs which can sensitize tumors cells to chemotherapy and radiation.
In this invention, we provide gene therapies for treating tumors, particularly schwannomas and related conditions. As a non-limiting example, direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. Gene therapies based on various AAV-P0-ICE vectors provide clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function. Our gene therapies effectively reduce the tumor size and pain associated with single schwannomas without causing the neurological damage typically associated with surgery. It is a much less invasive technique than surgery. Also, because our gene therapies do not deliver the gene that is mutated in the specific diseases but a pyroptotic gene, any Schwann-cell derived tumor, independent from its tumorigenic mutation, can be treated by the approaches described herein.